Methods of Reducing Repeat-Induced Silencing of Transgene Expression and Improved Fluorescent Biosensors

ABSTRACT

Methods of avoiding repeat- and homology-induced silencing of transgenes are disclosed, in which transgene sequences are genetically altered to reduce the affects of gene silencing. FRET biosensors containing such genetic alterations for improved expression in cell lines and in vivo are disclosed.

FIELD OF INVENTION

This invention relates to improved methods of expressing recombinantgenetic constructs in cells and whole organisms, and particularly to thedesign and expression of recombinant genetic constructs that exhibitreduced susceptibility to repeat- or homology-induced silencing oftransgene expression.

BACKGROUND OF INVENTION

Eukaryotic organisms possess a variety of efficient defense systems toguard against the invasion and expression of foreign nucleic acids.These defense systems have recently been recognized as a significanthurdle to gene therapy and other endeavors to express exogenoustransgenes in plants and animals. See, e.g., Bestor, 2000, Genesilencing as a threat to the success of gene therapy, J. Clin. Invest.105(4): 409-11. Although eukaryotic defense mechanisms may be mediatedby diverse modes of operation, one common trigger is the presence ofrepeat DNA in the transgene nucleic acid.

For instance, gene silencing may occur at either the transcriptional orpost-transcriptional level, and may accompany methylation of DNA andchanges in chromatin structure. One form of transcriptional silencinghas been termed “repeat-induced gene silencing” (RIGS), and wasdescribed at least thirteen years ago in Arabidopsis. Assaad et al.,1992, Somatic and germinal recombination of a direct repeat inArabidopsis, Genetics 132(2): 553-66. RIGS is strictly dependent on thepresence of repeated DNA sequences, and is correlated with the absenceof steady state mRNA, increased methylation of DNA and increasedresistance of DNA to enzymatic digestion. These observations led Ye andSigner to postulate that repeated nucleotide sequences lead chromatin toadopt a local configuration that is difficult to transcribe, similar toheterochromatin formation. Ye and Signer, 1996, RIGS (repeat-inducedgene silencing) in Arabidopsis is transcriptional and alters chromatinconfiguration.

More recently, RIGS has been described in other eukaryotic organisms andis now thought to be a universal silencing mechanism. Henikoff, 1998,Conspiracy of silence among repeated transgenes, Bioessays 20(7): 532-5.For instance, it has also been reported that DNA methylation and changesin chromatin structure are associated with RIGS in the fungus Neurosporacrassa. Meyer, 1996, Repeat-induced gene silencing: common mechanisms inplants and fungi, Biol. Chem. Hoppe Seyler 377(2): 87-95. Garrick andcolleagues reported that a reduction of transgene copy number intransgenic mouse lines resulted in a marked increase in transgeneexpression accompanied by decreased chromatin compaction and decreasedmethylation at the transgene locus. Garrick et al., 1998, Repeat-inducedgene silencing in mammals, Nat. Genet. 18(1): 5-6. In addition, it wasreported that inhibitors of histone deacetylase decrease the silencingof multicopy transgenes in murine embryonal carcinoma stem cells,suggesting that RIGS is at least one mechanism responsible fortriggering silencing in mammalian cells in vitro. McBurney et al., 2002,Evidence for repeat-induced gene silencing in cultured mammalian cells:inactivation of tandem repeats of transfected genes, Exp. Cell Res.274(1): 1-8. Although RIGS is associated with methylation in most cases,repeat transgenes are also subject to silencing in Drosophilamelanogaster, which exhibits no detectable modified DNA. Dorer andHenikoff, 1997, Transgene repeat arrays interact with distantheterochromatin and cause silencing in cis and trans, Genetics 147:1181-1190. Accordingly, methylation-independent mechanisms of RIGS mayalso exist.

RIGS has also been called “transcriptional cis-inactivation” in plantsbecause silencing is observed between neighboring repeated sequences andtransgene arrays. However, transcriptional gene silencing (TGS) oftransgenes can also occur in trans, both by a paramutation-likemechanism and by ectopic trans-inactivation. Vaucheret et al., 1998,Transgene-induced gene silencing in plants, Plant J. 16(6): 651.Paramutation is actually a natural epigenetic phenomenon where a hostgene can become silent and methylated when brought into the presence ofa silenced homologous copy, and can acquire the ability to inactivateother copies in subsequent crosses. Vaucheret at al., 1998; Meyer etal., 1993, Differences in DNA methylation are associated with aparamutation phenomenon in transgenic petunia, Plant J. 4:89-100. Themechanism is thought to involve DNA-DNA pairing and transmission ofchromatin structure from the silent copy to the inactive copy, as shownin Drosophila with the transmission of position-effect variegation(PEV). Vaucheret at al., 1998; Karpen, 1994, Position-effect variegationand the new biology of heterochromatin, Curr. Opin. Genetic Dev. 4:281-91.

Ectopic trans-inactivation differs from paramutation in that activetransgenes are silenced when brought into the presence of an unlinkedsilenced homologous transgene, but do not acquire the ability toinactivate in trans other unlinked transgenes. Vaucheret at al., 1998;Matzke et al., 1989, Reversible methylation and inactivation of markergenes in sequentially transformed tobacco plants, EMBO J. 8: 643-49.Deletion analysis has indicated that 90 base pairs of homology in thepromoter region of transgenes is sufficient for this type of silencing,indicating that homologous promoter regions may be one target for thisphenomenon. Thierry and Vaucheret, 1996, Sequence homology requirementsfor transcriptional silencing of 35S transgenes and post-transcriptionalsilencing of nitrate reductase (trans) genes by the tobacco 271 locus,Plant Mol. Biol. 32: 1075-83. Possible mechanisms for ectopictrans-inactivation include direct DNA pairing between a stablyintegrated transgene and another gene with a homologous promoter at aseparate location of the genome. Vaucheret, 1998. Another possiblemechanism could be the production of a diffusible RNA that leads tomethylation and silencing of the homologous locus via an RNA-DNAinteraction. Vaucheret at al., 1994, Promoter dependenttrans-inactivation in transgenic tobacco plants: kinetic aspects of genesilencing and gene reactivation, C.R. Acad. Sci. Paris 317:310-23; Parket al., 1996, Gene silencing mediated by promoter homology occurs at thelevel of transcription and results in meiotically heritable alterationsin methylation and gene activity, Plant J. 9: 183-94; Wassenegger andPelissier, 1998, A model for RNA-mediated gene silencing in higherplants, Plant Mol. Biol. 37: 349-62.

As noted above, gene silencing as a result of repeated DNA can alsooccur at the post-transcriptional level, i.e., when RNA does notaccumulate even in the presence of transcription. For instance, asreported by Ma and Mitra, transgenes with intrinsic direct repeatsinduced post-transcriptional gene silencing at a very high frequency intransgenic tobacco plants. Ma and Mitra, 2002, Intrinsic direct repeatsgenerate consistent post-transcriptional gene silencing in tobacco,Plant J. 31(1): 37-49. Others have shown that post-transcriptionalsilencing of nonviral transgenes in transgenic plants preventssubsequent virus infection when homology exists between transgene andviral sequences. English et al., 1996, Suppression of virus accumulationin transgenic plants exhibiting silencing of nuclear genes, Plant Cell8(2): 179-88. In the fungus N. crassa, repeat-induced point mutation(RIP) leads to both an increase in DNA methylation and degradation ofmRNA transcripts expressed from RIP regions. Galagan and Selker, 2004,RIP: the evolutionary cost of genome defense, Trends Genet. 20(9):417-23; Chicas et al., 2004, RNAi-dependent and RNAi-independentmechanisms contribute to the silencing of RIPed sequences in N. crassa,Nucleic Acids Res. 32(14): 4237-43.

Post-transcriptional gene silencing (PTGS) was originally discovered asthe coordinated silencing of transgenes and homologous host genes inplants, which was referred to as “co-suppression.” Napoli et al., 1990,Introduction of a chimeric chalcone synthesis gene into petunia resultsin reversible co-suppression of homologous genes in trans, Plant Cell2(4): 279-89. Since then, numerous transgenes encoding part or all ofthe entire transcribed sequence of a plant host gene have been shown totrigger co-suppression of homologous host genes. Depicker and vanMontagu, 1997, Post-transcriptional gene silencing in plants, Curr.Opinion Cell Biol. 9: 373-382. Co-suppression is commonly associatedwith strongly expressed transgenes, suggesting a mechanism related toaberrant levels of RNA or multiple gene copy number. See, e.g.,Lehtenberg et al., 2003, Neither inverted repeat T-DNA configurationsnor arrangements of tandemly repeated transgenes are sufficient totrigger transgene silencing, Plant J. 34(4): 507-17. However, PTGS ofhost gene expression has also been observed in the presence of weaklytranscribed or promoterless transgenes, implying that DNA-DNA pairingcould play a role in co-suppression. Vaucherot et al., 1998; vanBlokland et al., 1994, Transgene-mediated suppression of chalconesynthase expression in Petunia hybrida results in an increase in RNAturnover, Plant J. 6: 861-77.

More recently, a potent form of PTGS termed RNA interference (RNAi) hasbeen discovered. RNAi was first described in the invertebrate organismCaenorhabditis elegans, but is now known to occur in a wide variety ofeukaryotic organisms including fruit flies, zebra fish and mammals. Fireet al., 1998, Potent and specific genetic interference bydouble-stranded RNA in C. elegans, Nature 391: 806-11. The mechanism ofRNAi has been widely studied and involves the formation of a doublestranded RNA (dsRNA) with homology to a host gene, which is cleaved intosmall interfering RNA (siRNA) molecules that trigger the degradation ofhomologous host RNAs in the cytoplasm as wells as the de novomethylation of homologous DNA in the nucleus. Jana et al., 2004,Mechanisms and roles of the RNA-based gene silencing, Elec. J.Biotechnol. 7(3); Matzke and Birchler, 2005, RNAi-mediated pathways inthe nucleus, Nat. Rev. Genet. 6(1): 24-35.

Many researchers and companies have harnessed the specificity andpotency of RNAi to develop dsRNA-based therapeutics for silencingdisease genes and inhibiting virus expression and replication. However,very few researchers have focused on the obstacle that gene silencingmechanisms can present for gene therapy and expression of heterologousgenes in cells and whole organisms. U.S. Pat. No. 6,635,806 describesthe use of promoters, enhancers, coding sequences and terminators froman alternative plant species to avoid homology-based gene silencing intransgenic maize. US 20050191723 describes the use of StabilizingAnti-Repressor (STAR™) sequences for the expression of multipletransgenes. STAR™ sequences are described as DNA elements with genetranscription modulating activity that protect transgenes from genesilencing, and particularly RIGS. Finally, US 20031057715 describes theuse of low molecular weight, DNA-specific compounds that bind tochromatin-responsive elements (CRE), permitting chromatin remodeling andreduction of gene silencing in Drosophila. What is needed is auniversally applicable, straightforward method of improving transgenestructure to reduce or circumvent any repeat-driven gene silencingmechanism in any organism.

SUMMARY OF INVENTION

The present invention provides a solution to the interference by hostgene silencing mechanisms in the expression of homologous orheterologous genes or transgenes in a cell or whole organism. Inparticular, the present invention provides methods of reducing genesilencing of one or more transgenes in a cell, comprising introducing atleast one genetic alteration into said one or more transgenes such thatthe level of identity in at least one repeat or homologous region ofsaid one or more transgenes is reduced, and transfecting said one ormore transgenes into said cell, wherein gene silencing of said one ormore transgenes is there by reduced. The methods are applicable toreduce any type of gene silencing triggered by the presence of repeatDNA, including but not limited to repeat-induced gene silencing (RIGS),repeat-induced point mutation (RIP), paramutation, ectopictrans-inactivation, co-suppression and RNA interference. The methods arealso applicable where the repeat or homologous regions are present in asingle transgene, in two or more different transgenes, and where therepeat or homologous regions are present in both the transgene and theDNA of the host cell.

The methods of the present invention are applicable to a wide variety oftransgenes. For instance, the methods may be used in instances where thetransgene to be expressed exhibits a high level of identity with a hostgene, or where the transgene contains a domain or a stretch of basesexhibiting a high level of identity with a part of a host gene. Theinvention may be used to more efficiently express single transgenesencoding artificial single chain dimers produced by fusion of twomonomer sequences with a high level of identity. The methods may also beused to express single transgenes encoding proteins with duplicateddomains, e.g., ABC transporters, and for the expression of two or moredifferent transgenes encoding proteins with substantially similardomains.

In particular, the present inventors have found that the methods of thepresent invention are useful to increase the expression and efficacy ofligand binding fluorescent indicators, or biosensors, which comprise aligand binding protein moiety, a donor fluorophore moiety fused to theligand binding protein moiety, and an acceptor fluorophore moiety fusedto the ligand binding protein moiety. Because the two fluorophores ofmany biosensors are derived from the same fluorophore gene and exhibit ahigh level of identity, the present inventors have found that genesilencing may significantly affect the expression of such biosensors inwhole organisms and particularly plants. By reducing the identitybetween the fluorophore sequences of biosensors, the present inventorshave found that expression of such fluorophores may be significantlyenhanced.

Accordingly, in one embodiment, among others, the present inventionprovides an isolated nucleic acid which encodes a ligand bindingfluorescent indicator and methods of using the same, the indicatorcomprising a ligand binding protein moiety, a donor fluorophore moietyfused to the ligand binding protein moiety, and an acceptor fluorophoremoiety fused to the ligand binding protein moiety, wherein fluorescenceresonance energy transfer (FRET) between the donor moiety and theacceptor moiety is altered when the donor moiety is excited and saidligand binds to the ligand binding protein moiety, and wherein thenucleic acid sequence encoding at least one of either said donorfluorophore moiety or said acceptor fluorophore moiety has beengenetically altered to reduce the level of nucleic acid sequenceidentity between the nucleic acid encoding the donor fluorophore moietyand the nucleic acid encoding the acceptor fluorophore moiety. In themethods of the invention, either one or both of fluorophore sequencesmay be genetically altered to reduce the level of nucleic acid sequenceidentity.

A variety of genetic alterations may be used in the methods of theinvention, including but not limited to base changes encodingconservative amino acid substitutions and degenerate substitutions atwobble positions of the donor or acceptor fluorophore coding sequence.However, mutations that alter the emission or absorption spectra of thedonor and acceptor fluorophore moieties are excluded, as are alterationsthat adversely affect the activity of the biosensor.

Due to decreased interference from gene silencing, the biosensors of theinvention may demonstrate enhanced function in vivo upon expression ofthe genetically altered, encoding nucleic acid as compared to the sameor similar biosensor expressed from a nucleic acid not containing thegenetic alterations.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic drawing of a FLIP biosensor gene construct.

FIGS. 2A and 2B provide alignments showing the degree of homologybetween eCFP (SEQ ID NO: 1) and eYFP (SEQ ID NO: 2), and eCFP and eYFPVenus (SEQ ID NO: 3), respectively.

FIG. 3 is a diagram showing the FLIPgludelta13 construct used fortransformation of Arabidopsis.

FIG. 4 is a graph showing the change in fluorescence intensity over timein epidermal Arabidopsis cells of a five week old rdr6-11 plantexpressing FLIPglu600μdelta13 in response to glucose. +glc indicates theexternal application of 50 mM glucose. −glc indicates the removal ofexternal glucose. Perfusion was performed in NaPO₄ buffer, pH 7.

FIGS. 5A and 5B provide alignments showing the degree of homologybetween Ares (SEQ ID NO: 4) (genetically altered eCFP) and Aphrodite(SEQ ID NO: 5) (genetically altered Venus), and eCFP and Aphrodite,respectively. FIG. 5C provides an alignment showing the degree ofhomology between eCFP (SEQ ID NO: 1) and Mars (SEQ ID NO: 6)(genetically altered Venus).

FIG. 6 is a photograph showing transient expression ofFLIPglu600μdelta11 or delta13 in epidermal cells of Nicotianabenthamiana, and YFP fluorescence after excitation of YFP. A: eCFP andeYFP as FRET pair. B: delta11, with eCFP and Aphrodite encoding Venus asFRET pair. C: delta13, with eCFP and Aphrodite encoding Venus as FRETpair.

DETAILED DESCRIPTION Methods of Reducing Gene Silencing of Transgenes

As described above, the present invention provides methods of reducinggene silencing of one or more transgenes in a cell, comprisingintroducing at least one genetic alteration into said one or moretransgenes such that the level of identity or homology in at least onerepeat or homologous region of said one or more transgenes is reduced,and transfecting said one or more transgenes into said cell, whereingene silencing of said one or more transgenes is there by reduced.

As used herein, the phrase “gene silencing” is meant to encompass anyform of gene silencing, occurring at either the transcriptional orpost-transcriptional level, and including but not limited torepeat-induced gene silencing (RIGS), repeat-induced point mutation(RIP), paramutation, ectopic trans-inactivation, co-suppression and RNAinterference. Given that a common mechanism among different forms ofgene silencing is the presence of repeat or homologous regions of DNA,“gene silencing” may also be referred to as “repeat- or homology-inducedsilencing of gene expression or transgene expression,” or alternatively,“repeat- or homology-driven or -associated transgene silencing.” Thesealternative phrases are not to be confused with the specific phrase“repeat-induced gene silencing” or “RIGS,” which refers to a specifictype of transcriptional gene silencing involving changes in chromatinstructure and in some cases increased methylation. These alternativephrases are also not to be confused with “homology-induced genesilencing,” which is an art-recognized phrase used interchangeably withthe term “co-suppression,” i.e., where introduction of an exogenous geneshowing homology with an endogenous host gene leads topost-transcriptional gene silencing of both the exogenous and endogenousgene.

In the context of the present invention, the term “repeat” is used torefer to a sequence of DNA that is identical with another sequence ofDNA. The term “homology or “homologous” is used to refer to a sequenceof DNA having sufficient identity with another sequence of DNA so as toresult in a decrease in gene expression due to transcriptional orpost-transcriptional gene silencing. Such regions may be present withina single transgene, in one or more transgenes, or in one or moretransgenes when compared to the host genome. The presence of suchregions in a transgene may be detected by an increase in transgeneexpression when the transgene is expressed in a host cell that isdeficient in one or more forms of gene silencing as described herein.

“Repeat” and “homologous” regions according to the invention may be anylength that is sufficient to result in gene silencing, but are typicallyat least 10, at least 15, at least 20, at least 25, at least 30, atleast 40, at least 50, at least 75, at least 100 or at least 200 basesin length. “Homologous” regions are at least 50%, at least 60%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 98%, or at least 99% identical. Since gene silencing insome cases involves small double-stranded RNAs derived from therespective gene with sizes ranging between 21 and 28 base pairs, arepeat in such embodiments includes sequences of at least 21 bases withup to three mismatches in the preferred case, or up to two mismatches ina less preferred case or one mismatch in a less preferred case.

“Gene silencing” is meant to refer to any decrease in the level of geneexpression, or the level of RNA or protein produced from an expressedgene, as a result of the presence of repeat or homologous regions ofDNA. As such, methods of “reducing” or “decreasing” gene silencing aremeant to refer to any method in which gene silencing is reduced ordecreased but not necessarily eliminated or inhibited. Methods ofeliminating or inhibiting gene silencing using the methods describedherein are also included. A decrease in gene silencing may be detectedby measuring mRNA levels or protein levels resulting from the disclosedmethods of the invention as compared to mRNA or protein levels in thesame host cell or organism in the absence of the methods of theinvention.

As used herein, the term “transgene” refers to any isolated “exogenous”gene to be expressed recombinantly in a host cell or whole organism, incontrast to “endogenous” genes that are expressed from the host cellgenome. Transgenes include “heterologous” genes, which are genes fromthe genome of one organism that are placed into a different organism orcell of a different organism. Transgenes also include exogenous genesoriginating from the same organism as the host cell or host organism,for instance, that have been mutated or placed under differentregulatory sequences than the endogenous gene such that they take on adifferent function or expression characteristic. It is also possible tointroduce an exogenous gene originating from the host cell or organisminto the host for the purpose of complementing a defective endogenousgene or increasing the copy number or expression level of a similarendogenous gene. The term “gene” is meant to include not only theprotein coding portion of a nucleic acid, but also the promoter regionand any upstream and downstream regulatory regions involved inexpression of the gene, including transcription and translation.

The methods of the invention include the use of any genetic alterationto a repeat or homologous region of a gene involved in gene silencingwith the purpose of reducing gene silencing and increasing geneexpression, including but not limited to substitutions, insertions anddeletions, so long as the genetic alteration reduces gene silencing,increases gene expression, and does not adversely affect the function ofthe protein encoded by the transgene. Such alterations include geneticmodifications of the upstream and downstream regulatory regions of atransgene. Such alterations also include those encoding conservativeamino acid substitutions in the transgene coding sequence.

Conservative amino acid substitutions are generally defined as aminoacid replacements that preserve the structure and functional propertiesof proteins. The chemical properties of amino acids that permit one tobe conservatively substituted for another are well known by those ofskill in the art. For instance, hydrophobic amino acids includemethionine, alanine, valine, leucine, isoleucine and norleucine. Neutraland hydrophilic amino acids include cysteine, serine and threonine.Acidic amino acids include aspartate and glutamate. Basic amino acidsinclude asparagine, glutamine, histidine, lysine and arginine. Aromaticamino acids include tryptophan, tyrosine and phenylalanine. Glycine andproline are two amino acids that can influence chain orientation andbending.

In one embodiment of the invention, degenerate substitutions may be madeat one or more wobble positions of the transgene. Such substitutions arepreferred because they change the nucleic acid coding sequence of thetransgene without changing the encoded amino acid sequence. The term“wobble” is an art-recognized term that refers to reduced constraint ata position of an anticodon of tRNA that allows alignment of the tRNAwith several possible codons. This redundancy is typically seen at thethird codon position, for example, both GAA and GAG code for the aminoacid glutamine. This property of the genetic code makes it more tolerantof mutations. For instance, four-fold degenerate codons can tolerate anymutation at the third position. Two-fold degenerate codons can tolerateone out of the three base substitutions at the third position. Thefollowing table shows the most popular twenty amino acids and the codonsthat code for each amino acid.

TABLE 1 Amino Acids and Corresponding Codons Amino Acid AbbreviationCorresponding Codons Alanine A GCU, GCC, GCG, GCA Arginine R AGA, AGG,CGU, CGG, CGC, CGA Asparagine N AAU, AAC Aspartic Acid D GAU, GACCysteine C UGU, UGC Glutamine Q CAA, CAG Glutamic Acid E GAA, GAGGlycine G GGU, GGC, GGA, GGG Histidine H CAU, CAC Isoleucine I AUU, AUC,AUA Leucine L UUA, UUG, CUU, CUC, CUG, CUA Lysine K AAA, AAG MethionineM AUG Phenylalanine F UUU, UUC Proline P CCU, CCA, CCC, CCG Serine SAGU, AGC, UCC, UCU, UCA, UCG Threonine T ACU, ACA, ACC, ACG Tryptophan WUGG Tyrosine Y UAU, UAC Valine V GUG, GUC, GUA, GUU Start AUG, GUG StopUAG, UGA, UAA

In the methods of the present invention, any number of geneticalterations may be made in a transgene in order to alter the level ofidentity between repeat or homologous sequences. Where repeat orhomologous sequences exist between two transgenes, different alterationsmay be made in each transgene sequence to further decrease the level ofidentity between the two sequences. For instance, in the methods of theinvention, at least two, at least five, at least ten, at least fifteen,at least twenty, at least thirty, at least fifty, or at least onehundred degenerate substitutions may be made at the wobble positions ofeach transgene involved in the gene silencing.

In designing genetic substitutions for the methods of the presentinvention, the skilled artisan may chose to consider any codon biaspresent in the host cell or organism in order to further optimizeexpression. For example, G and C ending codons have been found to bemost prevalent in monocot plant species as well as Drosophila. Kawabeand Miyashita, 2003, Patterns of codon usage bias in three dicot andfour monocot plant species, Genes Genet. Syst. 78(5): 343-52. InArabidopsis, codon usage has been associated with gene function, withG/C biased codon usage seen in photosynthetic and housekeeping genes,and A/T biased codon usage found in tissue-specific and stress-inducedgenes. Chiapello et al., 1998, Codon usage and gene function are relatedin sequences of Arabidopsis thaliana, Gene 209(1-2): GC1-GC38. Inhumans, codon usage preference has been shown to vary according todistance from RNA splice sites. Willie and Majewski, 2004, Evidence forcodon bias selection at the pre-mRNA level in eukaryotes, Trends Genet.20(11): 534-38. And organisms with a high metabolic rate contain proteinencoding genes with more A-ending codons and have a higher A content intheir introns than do organisms with a low metabolic rate. Xia, 1996,Maximizing transcription efficiency causes codon usage bias, Genetics144(3): 1309-20.

As described above, preferred genetic alterations will result in amodified coding sequence but no changes in amino acid sequence. Wheregenetic alterations do produce a transgenic protein having one or moreconservative substitutions, or insertions or deletions that do notadversely affect protein function, such isolated proteins are alsoincluded in the present invention. Vectors, prokaryotic and eukaryotichost cells and transgenic organisms comprising the improved nucleicacids of the invention are also included.

The methods of the present invention will find use in a wide variety ofeukaryotic cells and organisms where gene silencing results is areduction in transgene expression, including plants, animals and fungi.For instance, the methods of the invention may be used to express singletransgenes in cells and organisms containing one or more host genes withregions containing repeat or homologous regions as compared to thetransgene sequence, or in methods of expressing two or more transgenesfrom the same or different construct having regions of sequencesimilarity, e.g., two members of the same gene family. The methods ofthe invention may be used to reduce gene silencing of single transgenesencoding artificial single chain dimers, e.g., single chain hormones orother glycoproteins that naturally exist as homodimers but have beenrecombinantly fused perhaps with the intent of introducing a functionalmutation in one of the monomers. The methods of the present inventionmay also be used for the expression of transgenes encoding proteins withduplicated domains, for example, ABC transporters (van der Heide andPoolman, 2002, ABC transporters: one, two or four extracytoplasmicsubstrate binding sites, EMBO Rep. 3(10): 938-43), beta-propellerdomain/kelch repeat-containing proteins (Prag and Adams, 2003, Molecularphylogeny of the kelch-repeat superfamily reveals an expansion ofBTB/kelch proteins in animals, BMC Bioinformatics 4: 42), andthrombospondin repeat-containing proteins to name a few (Meiniel et al.,2003, The thrombospondin type 1 repeat (TSR) and neuronaldifferentiation: roles of SCO-spondin oligopeptides on neuronal celltypes and cell lines, Int. Rev. Cytol. 230: 1-39).

In one embodiment, the methods of the invention may be used to enhancethe expression of biosensor transgenes in a host cell or organism, aswell as the simultaneous expression of more than one fluorescentbiosensor in one cell. More broadly, the methods of the invention mayalso be employed with any use of FRET employing GFP variants, forexample in the detection of protein interactions.

Biosensors

As mentioned above, the present inventors have surprisingly found thatthe methods of the present invention are useful to increase theexpression and efficacy of ligand binding fluorescent indicators, orFRET-based biosensors. Exemplary biosensors are described in provisionalapplication Ser. No. 60/643,576, provisional application Ser. No.60/658,141, provisional application Ser. No. 60/658,142, provisionalapplication Ser. No. 60/657,702, PCT application [Attorney Docket No.056100-5053, “Phosphate Biosensors and Methods of Using the Same”], andPCT application [Attorney Docket No. 056100-5055, “Sucrose Biosensorsand Methods of Using the Same], which are herein incorporated byreference in their entireties. Such biosensors comprise a ligand bindingprotein moiety, a donor fluorophore moiety fused to the ligand bindingprotein moiety, and an acceptor fluorophore moiety fused to the ligandbinding protein moiety. Because the two fluorophores of many biosensorsare derived from the same fluorophore gene and exhibit a high level ofidentity, the present inventors have found that gene silencing maysignificantly affect the expression of such biosensors in wholeorganisms and particularly plants. By reducing the identity between thefluorophore sequences of biosensors, the present inventors have foundthat expression of the biosensors in a cell or organism may besignificantly enhanced.

Accordingly, in one embodiment, among others, the present inventionprovides an isolated nucleic acid which encodes a ligand bindingfluorescent indicator and methods of using the same, the indicatorcomprising a ligand binding protein moiety, a donor fluorophore moietyfused to the ligand binding protein moiety, and an acceptor fluorophoremoiety fused to the ligand binding protein moiety, wherein fluorescenceresonance energy transfer (FRET) between the donor moiety and theacceptor moiety is altered when the donor moiety is excited and saidligand binds to the ligand binding protein moiety, and wherein thenucleic acid sequence encoding at least one of either said donorfluorophore moiety or said acceptor fluorophore moiety has beengenetically altered to reduce the level of nucleic acid sequenceidentity between the nucleic acid encoding the donor fluorophore moietyand the nucleic acid encoding the acceptor fluorophore moiety in orderto reduce gene silencing of the indicator transgene.

In the methods of the invention, either one or both of fluorophoresequences may be genetically altered to reduce the level of nucleic acidsequence identity. The fluorophore coding sequences may be fused to thetermini of the ligand binding domain. Alternatively, either or both ofthe donor fluorophore and/or said acceptor fluorophore moieties may befused to the ligand binding protein moiety at an internal site of saidligand binding protein moiety. Such fusions are described in provisionalapplication No. 60/658,141, which is herein incorporated by reference.Preferably, the donor and acceptor moieties are not fused in tandem,although the donor and acceptor moieties may be contained on the sameprotein domain or lobe. A domain is a portion of a protein that performsa particular function and is typically at least about 40 to about 50amino acids in length. There may be several protein domains contained ina single protein.

A “ligand binding protein moiety” according to the present invention canbe a complete, naturally occurring protein sequence, or at least theligand binding portion or portions thereof. In preferred embodiments,among others, a ligand binding moiety of the invention is at least about40 to about 50 amino acids in length, or at least about 50 to about 100amino acids in length, or more than about 100 amino acids in length.

Preferred ligand binding protein moieties according to the presentinvention, among others, are transporter proteins and ligand bindingsequences thereof, for instance transporters selected from the groupconsisting of channels, uniporters, coporters and antiporters. Alsopreferred are periplasmic binding proteins (PBP), such as any of thebacterial PBPs included in Table 2 below. Bacterial PBPs comprise twoglobular domains or lobes and are convenient scaffolds for designingFRET sensors. Fehr et al., 2003, J. Biol. Chem. 278: 19127-33. Thebinding site is located in the cleft between the domains, and uponbinding, the two domains engulf the substrate and undergo a hinge-twistmotion. Quiocho and Ledvina, 1996, Mol. Microbiol. 20: 17-25. In type IPBPs, such as GGBP (D-GalactoseD-Glucose Binding Protein), the terminiare located at the proximal ends of the two lobes that move apart uponligand binding. Fehr et al., 2004, Current Opinion in Plant Biology 7:345-51. In type II PBPs, such as Maltose Binding Protein (MBP), thetermini are located at the distal ends of the lobes relative to thehinge region and come closer together upon ligand binding. Thus,depending on the type of PBP and/or the position of the fused donor oracceptor moiety, FRET may increase or decrease upon ligand binding andboth instances are included in the present invention.

TABLE 2 Bacterial Periplasmic Binding Proteins Gene name SubstrateSpecies 3D Reference AccA agrocinopine Agrobacterium sp. —/— J.Bacteriol. (1997) 179, 7559-7572 AgpE alpha-glucosides (sucrose,maltose, Rhizobium meliloti —/— J. Bacteriol. (1999) 181, 4176-4184trehalose) AlgQ2 Alginate Sphingomonassp. —/c J. Biol. Chem. (2003) 278,6552-6559 AlsB Allose E. coli —/c J. Bacteriol. (1997) 179, 7631-7637 J.Mol. Biol. (1999) 286, 1519-1531 AraF Arabinose E. coli —/c J. Mol.Biol. (1987) 197, 37-46 J. Biol. Chem. (1981) 256, 13213-13217 AraSArabinose/fructose/xylose Sulfolobus solfataricus —/— Mol. Microbiol.(2001) 39, 1494-1503 ArgT lysine/arginine/ornithine Salmonellatyphimurium o/c Proc. Natl. Acad. Sci. USA (1981) 78, 6038-6042 J. Biol.Chem. (1993) 268, 11348-11355 ArtI Arginine E. coli Mol. Microbiol.(1995) 17, 675-686 ArtJ Arginine E. coli Mol. Microbiol. (1995) 17,675-686 b1310 (putative, multiple sugar) E. coli —/— NCBI accessionA64880 b1487 (putative, oligopeptide binding) E. coli —/— NCBI accessionB64902 b1516 (sugar binding protein homolog) E. coli —/— NCBI accessionG64905 BtuF vitamin B12 E. coli —/— J. Bacteriol. (1986) 167, 928-934CAC1474 proline/glycine/betaine Clostridium acetobutylicum —/— NCBIaccession AAK79442 Cbt dicarboxylate E. coli —/— J. Supramol. Struct.(1977) 7, 463-80 (succinate, malate, fumarat) J. Biol. Chem. (1978) 253,7826-7831 J. Biol. Chem. (1975) 250, 1600-1602 CbtA CellobioseSulfoblobus solfataricus —/— Mol. Microbiol. (2001) 39, 1494-1503 ChvESugar Agrobacterium —/— J. Bacteriol. (1990) 172, 1814-1822 tumefaciensCysP thiosulfate E. coli —/— J. Bacteriol. (1990) 172, 3358-3366 DctPC4-dicarboxylate Rhodobacter capsulatus —/— Mol. Microbiol. (1991) 5,3055-3062 DppA dipeptides E. coli o/c Biochemistry (1995) 34,16585-16595 FbpA Iron Neisseria gonorrhoeae —/c J. Bacteriol. (1996)178, 2145-2149 FecB Fe(III)-dicitrate E. coli J. Bacteriol. (1989) 171,2626-2633 FepB enterobactin-Fe E. coli —/— J. Bacteriol. (1989) 171,5443-5451 Microbiology (1995) 141, 1647-1654 FhuD ferrichydroxamate E.coli —/c Mol. Gen. Genet. (1987) 209, 49-55 Nat. Struct. Biol. (2000) 7,287-291 Mol. Gen. Genet. (1987) 209, 49-55 FliY Cystine E. coli —/— J.Bacteriol. (1996) 178, 24-34 NCBI accession P39174 GlcSglucose/galactose/mannose Sulfolobus solfataricus —/— Mol. Microbiol.(2001) 39, 1494-1503 GlnH Glutamine E. coli o/— Mol. Gen. Genet. (1986)205, 260-9 (protein: J. Mol. Biol. (1996) 262, 225-242 GLNBP) J. Mol.Biol. (1998) 278, 219-229 GntX Gluconate E. coli —/— J. Basic.Microbiol. (1998) 38, 395-404 HemT Haemin Yersinia enterocolitica —/—Mol. Microbiol. (1994) 13, 719-732 HisJ Histidine E. coli —/cBiochemistry (1994) 33, 4769-4779 (protein: HBP) HitA Iron Haemophilusinfluenzae o/c Nat. Struct. Biol. (1997) 4, 919-924 Infect. Immun.(1994) 62, 4515-25 J. Biol. Chem. (195) 270, 25142-25149 LivJleucine/valine/isoleucine E. coli —/c J. Biol. Chem. (1985) 260,8257-8261 J. Mol. Biol. (1989) 206, 171-191 LivK Leucine E. coli —/c J.Biol. Chem. (1985) 260, 8257-8261 (protein: L- J. Mol. Biol. (1989) 206,193-207 BP) MalE maltodextrine/maltose E. coli o/c Structure (1997) 5,997-1015 (protein: J. Bio.l Chem. (1984) 259, 10606-13 MBP) MglBglucose/galactose E. coli —/c J. Mol. Biol. (1979) 133, 181-184(protein: Mol. Gen. Genet. (1991) 229, 453-459 GGBP) ModA molybdate E.coli —/c Nat. Struct. Biol. (1997) 4, 703-707 Microbiol. Res. (1995)150, 347-361 MppA L-alanyl-gamma-D-glutamyl-meso- E. coli J. Bacteriol.(1998) 180, 1215-1223 diaminopimelate NasF nitrate/nitrite Klebsiellaoxytoca —/— J. Bacteriol. (1998) 180, 1311-1322 NikA Nickel E. coli —/—Mol. Microbiol. (1993) 9, 1181-1191 opBC Choline Bacillus subtilis —/—Mol. Microbiol. (1999) 32, 203-216 OppA oligopeptide Salmonellatyphimurium o/c Biochemistry (1997) 36, 9747-9758 Eur. J. Biochem.(1986) 158, 561-567 PhnD alkylphosphonate E. coli —/— J. Biol. Chem.(1990) 265, 4461-4471 PhoS (Psts) phosphate E. coli —/c J. Bacteriol.(1984) 157, 772-778 Nat. Struct. Biol. (1997) 4, 519-522 PotDputrescine/spermidine E. coli —/c J. Biol. Chem. (1996) 271, 9519-9525PotF polyamines E. coli —/c J. Biol. Chem. (1998) 273, 17604-17609 ProXBetaine E. coli J. Biol. Chem. (1987) 262, 11841-11846 rbsB Ribose E.coli o/c J. Biol. Chem. (1983) 258, 12952-6 J. Mol. Biol. (1998) 279,651-664 J. Mol. Biol. (1992) 225, 155-175 SapA Peptides Salmonellatyphimurium —/— EMBO J. (1993) 12, 4053-4062 Sbp Sulfate Salmonellatyphimurium —/c J. Biol. Chem. (1980) 255, 4614-4618 Nature (1985) 314,257-260 TauA Taurin E. coli —/— J. Bacteriol. (1996) 178, 5438-5446 TbpAThiamin E. coli —/— J. Biol. Chem. (1998) 273, 8946-8950 TctCtricarboxylate Salmonella typhimurium —/— ThuE Trehalose/maltose/sucroseSinorhizobium meliloti —/— J. Bacteriol. (2002) 184, 2978-2986 TreSTrehalose Sulfolobus solfataricus —/— Mol. Microbiol. (2001) 39,1494-1503 tTroA Zinc Treponema pallidum —/c Gene (1997) 197, 47-64 Nat.Struct. Biol. (1999) 6, 628-633 UgpB sn-glycerol-3-phosphate E. coli —/—Mol. Microbiol. (1988) 2, 767-775 XylF Xylose E. coli —/— ReceptorsChannels (1995) 3, 117-128 YaeC Unknown E. coli —/— J Bacteriol (1992)174, 8016-22 NCBI accession P28635 YbeJ (GltI) Glutamate/aspartate(putative, E. coli —/— NCBI accession E64800 superfamily:lysine-arginine-ornithine- binding protein) YdcS (putative, spermidine)E. coli —/— NCBI accession P76108 (b1440) YehZ Unknown E. coli —/— NCBIaccession AE000302 YejA (putative, homology to periplasmic E. coli —/—NCBI accession AAA16375 oligopeptide-binding protein - Helicobacterpylori) YgiS oligopeptides E. coli —/— NCBI accession Q46863 (b3020)YhbN Unknown E. coli —/— NCBI accession P38685 YhdW (putative, aminoacids) E. coli —/— NCBI accession AAC76300 YliB (b0830) (putative,peptides) E. coli —/— NCBI accession P75797 YphF (putative sugars) E.coli —/— NCBI accession P77269 Ytrf Acetoin B. subtilis —/— J.Bacteriol. (2000) 182, 5454-5461 ZnuA Zinc Synechocystis —/— J. Mol.Biol. (2003) 333, 1061-1069

Bacterial PBPs have the ability to bind a variety of different moleculesand nutrients, including sugars, amino acids, vitamins, minerals, ions,metals and peptides, as shown in Table 2. Thus, PBP-based ligand bindingsensors may be designed to permit detection and quantitation of any ofthese molecules according to the methods of the present invention.Naturally occurring species variants of the PBPs listed in Table 2 mayalso be used, in addition to artificially engineered variants comprisingsite-specific mutations, deletions or insertions that maintainmeasurable ligand binding function. Variant nucleic acid sequencessuitable for use in the nucleic acid constructs of the present inventionwill preferably have at least 70, 75, 80, 85, 90, 95, or 99% similarityor identity to the native gene sequence for a given PBP.

Suitable variant nucleic acid sequences may also hybridize to the genefor a PBP under highly stringent hybridization conditions. Highstringency conditions are known in the art; see for example Maniatis etal., Molecular Cloning: A Laboratory Manual, 2d Edition, 1989, and ShortProtocols in Molecular Biology, ed. Ausubel, et al., both of which arehereby incorporated by reference. Stringent conditions aresequence-dependent and will be different in different circumstances.Longer sequences hybridize specifically at higher temperatures. Anextensive guide to the hybridization of nucleic acids is found inTijssen, Techniques in Biochemistry and Molecular Biology—Hybridizationwith Nucleic Acid Probes, “Overview of principles of hybridization andthe strategy of nucleic acid assays” (1993). Generally, stringentconditions are selected to be about 5-10° C. lower than the thermalmelting point (Tm) for the specific sequence at a defined ionic strengthand pH. The Tm is the temperature (under defined ionic strength, pH andnucleic acid concentration) at which 50% of the probes complementary tothe target hybridize to the target sequence at equilibrium (as thetarget sequences are present in excess, at Tm, 50% of the probes areoccupied at equilibrium). Stringent conditions will be those in whichthe salt concentration is less than about 1.0M sodium ion, typicallyabout 0.01 to 1.0M sodium ion concentration (or other salts) at pH 7.0to 8.3 and the temperature is at least about 30° C. for short probes(e.g. 10 to 50 nucleotides) and at least about 60° C. for long probes(e.g. greater than 50 nucleotides). Stringent conditions may also beachieved with the addition of destabilizing agents such as formamide.

Preferred artificial variants of the sensors of the present inventionmay exhibit increased or decreased affinity for ligands, in order toexpand the range of ligand concentration that can be measured.Artificial variants showing decreased or increased binding affinity forglutamate may) be constructed by random or site-directed mutagenesis andother known mutagenesis techniques, and cloned into the vectorsdescribed herein and screened for activity according to the disclosedassays.

In the biosensor nucleic acids of the present invention, fluorescentdomains can optionally be separated from the ligand binding domain byone or more flexible linker sequences. Such linker moieties arepreferably between about 1 and 50 amino acid residues in length, andmore preferably between about 1 and 30 amino acid residues. Linkermoieties and their applications are well known in the art and described,for example, in U.S. Pat. Nos. 5,998,204 and 5,981,200, and Newton etal., Biochemistry 35:545-553 (1996). Alternatively, shortened versionsof the fluorophores or the binding proteins described herein may beused.

For instance, the present inventors have also found that removingsequences connecting the core protein structure of the binding domainand the fluorophore, i.e., by removing linker sequences and/or bydeleting amino acids from the ends of the analyte binding moiety and/orthe fluorophores, closer coupling of fluorophores is achieved leading tohigher ratio changes. Preferably, deletions are made by deleting atleast one, or at least two, or at least three, or at least four, or atleast five, or at least eight, or at least ten, or at least fifteennucleotides in a nucleic acid construct encoding a FRET biosensor thatare located in the regions encoding the linker, or fluorophore, orligand binding domains. Deletions in different regions may be combinedin a single construct to create more than one region demonstratingincreased rigidity. Amino acids may also be added or mutated to increaserigidity of the biosensor and improve sensitivity. For instance, byintroducing a kink by adding a proline residue or other suitable aminoacid. Improved sensitivity may be measured by the ratio change in FRETfluorescence upon ligand binding, and preferably increases by at least afactor of 2 as a result of said deletion(s). See provisional applicationNo. 60/658,141, which is herein incorporated by reference in itsentirety.

The isolated nucleic acids of the invention may incorporate any suitabledonor and acceptor fluorescent protein moieties that are capable incombination of serving as donor and acceptor moieties in FRET. Preferreddonor and acceptor moieties are selected from the group consisting ofGFP (green fluorescent protein), CFP (cyan fluorescent protein), BFP(blue fluorescent protein), YFP (yellow fluorescent protein), andenhanced variants thereof, with a particularly preferred embodimentprovided by the donor/acceptor pair CFP/YFP-Venus, a variant of YFP withimproved pH tolerance and maturation time (Nagai, T., Ibata, K., Park,E. S., Kubota, M., Mikoshiba, K., and Miyawaki, A. (2002) A variant ofyellow fluorescent protein with fast and efficient maturation forcell-biological applications. Nat. Biotechnol. 20, 87-90). Analternative is the MiCy/mKO pair with higher pH stability and a largerspectral separation (Karasawa S, Araki T, Nagai T, Mizuno H, Miyawaki A.Cyan-emitting and orange-emitting fluorescent proteins as adonor/acceptor pair for fluorescence resonance energy transfer. BiochemJ. 2004 381:307-12). Also suitable as either a donor or acceptor isnative DsRed from a Discosoma species, an ortholog of DsRed from anothergenus, or a variant of a native DsRed with optimized properties (e.g. aK83M variant or DsRed2 (available from Clontech)). Criteria to considerwhen selecting donor and acceptor fluorescent moieties are known in theart, for instance as disclosed in U.S. Pat. No. 6,197,928, which isherein incorporated by reference in its entirety.

As used herein, the term “fluorophore variant” is intended to refer topolypeptides with at least about 70%, more preferably at least 75%identity, including at least 80%, 90%, 95% or greater identity to nativefluorescent molecules. Many such variants are known in the art, or canbe readily prepared by random or directed mutagenesis of nativefluorescent molecules (see, for example, Fradkov et al., FEBS Lett.479:127-130 (2000)).

The invention further provides vectors containing isolated nucleic acidmolecules encoding the improved biosensor genes as disclosed herein.Exemplary vectors include vectors derived from a virus, such as abacteriophage, a baculovirus or a retrovirus, and vectors derived frombacteria or a combination of bacterial sequences and sequences fromother organisms, such as a cosmid or a plasmid. Vectors may be adaptedfor function in a prokaryotic cell, such as E. coli or other bacteria,or a eukaryotic cell, including yeast, plant and animal cells. Forinstance, the vectors of the invention will generally contain elementssuch as an origin of replication compatible with the intended hostcells, one or more selectable markers compatible with the intended hostcells and one or more multiple cloning sites. The choice of particularelements to include in a vector will depend on factors such as theintended host cells, the insert size, whether) regulated expression ofthe inserted sequence is desired, i.e., for instance through the use ofan inducible or regulatable promoter, the desired copy number of thevector, the desired selection system, and the like. The factors involvedin ensuring compatibility between a host cell and a vector for differentapplications are well known in the art.

Preferred vectors for use in the present invention will permit cloningof the ligand binding domain or receptor genetically fused to nucleicacids encoding donor and acceptor fluorescent molecules, resulting inexpression of a chimeric or fusion protein comprising the ligand bindingdomain genetically fused to donor and acceptor fluorescent molecules.Exemplary vectors include the bacterial pRSET-FLIP derivatives disclosedin Fehr et al. (2002) (Visualization of maltose uptake in living yeastcells by fluorescent nanosensors. Proc. Natl. Acad. Sci. USA 99,9846-9851), which is herein incorporated by reference in its entirety.Methods of cloning nucleic acids into vectors in the correct frame so asto express fusion proteins are well known in the art.

The invention also includes host cells transfected with a vector or anexpression vector of the invention, including prokaryotic cells, such asE. coli or other bacteria, or eukaryotic cells, such as yeast cells,plant cells or animal cells. In another aspect, the invention features atransgenic non-human animal having a phenotype characterized byexpression of the nucleic acid sequence coding for the expression of thebiosensor. The phenotype is conferred by a transgene contained in thesomatic and germ cells of the animal, which may be produced by (a)introducing a transgene into a zygote of an animal, the transgenecomprising a DNA construct encoding the biosensor; (b) transplanting thezygote into a pseudopregnant animal; (c) allowing the zygote to developto term; and (d) identifying at least one transgenic offspringcontaining the transgene. The step of introducing of the transgene intothe embryo can be by introducing an embryonic) stem cell containing thetransgene into the embryo, or infecting the embryo with a retroviruscontaining the transgene. Transgenic animals of the invention includetransgenic C. elegans and transgenic mice and other animals.

Transgenic plants expressing the nucleic acids described herein are alsoincluded in the present invention. Transgenic crops include, forexample, tobacco, sugar beet, soy beans, beans, peas, potatoes, rice ormaize. The expression of genes in dicotyledonous and monocotyledonousplants can be achieved by a variety of procedures known and routinelyapplied. See, e.g., Potrykus, 1990, Gene transfer methods for plants andcell cultures, Ciba Found. Symp. 154: 198-208. One example istransformation of plants cells with a T-DNA containing the gene ofinterest using Agrobacterium tumefaciens or Agrobacterium rhizogenes asa means of transformation. For the use of Agrobacterium for theintroduction of a gene into a plant cell, the respective gene should becloned into a binary vector. A variety of different cloning vectors isavailable for) expression of genes in higher plants using Agrobacterium,e.g. mini binary vectors (Xiang et al., 1999: a mini binary vectorseries for plant transformation, Plant. Mol. Biol. 40(4): 711-7) andvectors of the pPZP series (Hajdukiewicz et al., 1994, The small,versatile pPZP family of Agrobacterium binary vectors for planttransformation, Plant. Mol. Biol. 25(6): 989-94). Binary planttransformation vectors can replicate in E. coli as well as inAgrobacterium and contain selection markes for selection of transformedplants. For the transfer of the T-DNA, infection of the plant byAgrobacterium is necessary; this can be by infection of leaf pieces,roots, protoplasts, suspension cultures, or flowers of whole plants. Forthe transformation of Arabidopsis plants, a dipping method is mostcommonly used (Clough and Bent, 1998, Floral dip: a simplified methodfor Agrobacterium-mediated transformation of Arabidopsis thaliana, PlantJ. 16(6): 735-43). Transformed plants are then selected for resistanceagainst the selection marker, e.g. kanamycin, hygromycin, gluphosinate.

Besides transformation using Agrobacteria, there are many othertechniques available for the expression of genes in a plant host cell.These techniques include the fusion or transformation of protoplasts,microinjection of DNA and electroporation, as well as ballistic methodsand virus infection. From the transformed plant material, whole plantscan be regenerated in a suitable medium, which contains antibiotics orbiocides for selection. No special demands are required for plasmidinjection and electroporation. Simple plasmids, such as, e.g.,pUC-derivatives can be used. Should, however, whole plants beregenerated from such transformed cells, the presence of a selectablemarker gene is necessary.

The present invention also encompasses isolated biosensor moleculeshaving the properties described herein, particularly PBP-basedfluorescent indicators. Such polypeptides are preferably recombinantlyexpressed using the nucleic acid constructs described herein. The)expressed polypeptides can optionally be produced in and/or isolatedfrom a transcription-translation system or from a recombinant cell, bybiochemical and/or immunological purification methods known in the art.The polypeptides of the invention can be introduced into a lipidbilayer, such as a cellular membrane extract, or an artificial lipidbilayer (e.g. a liposome vesicle) or nanoparticle.

The present invention includes methods of detecting changes in thelevels of ligands in samples, comprising (a) providing a cell expressinga nucleic acid encoding an improved sensor according to the presentinvention and a sample comprising said ligand; and (b) detecting achange in FRET between said donor fluorescent protein moiety and saidacceptor fluorescent protein moiety, wherein a change in FRET betweensaid donor moiety and said acceptor moiety indicates a change in thelevel of said ligand in the sample. The ligand may be any suitableligand for which a fused FRET biosensor may be constructed, includingany of the ligands described herein. Preferably the ligand is onerecognized by a PBP, and more preferably a bacterial PBP, such as thoseincluded in Table 2 and homologues and natural and artificial variantsthereof.

FRET may be measured using a variety of techniques known in the art. Forinstance, the step of determining FRET may comprise measuring lightemitted from the acceptor fluorescent protein moiety. Alternatively, thestep of determining FRET may comprise measuring light emitted from thedonor fluorescent protein moiety, measuring light emitted from theacceptor fluorescent protein moiety, and calculating a ratio of thelight emitted from the donor fluorescent protein moiety and the lightemitted from the acceptor fluorescent protein moiety. The step ofdetermining FRET may also comprise measuring the excited state lifetimeof the donor moiety or) anisotropy changes (Squire A, Verveer P J, RocksO, Bastiaens P I. J Struct Biol. 2004 July; 147(1):62-9. Red-edgeanisotropy microscopy enables dynamic imaging of homo-FRET between greenfluorescent proteins in cells.). Such methods are known in the art anddescribed generally in U.S. Pat. No. 6,197,928, which is hereinincorporated by reference in its entirety.

The amount of ligand in a sample can be determined by determining thedegree of FRET. First the sensor must be introduced into the sample.Changes in ligand concentration can be determined by monitoring FRETchanges at time intervals. The amount of ligand in the sample can bequantified for example by using a calibration curve established bytitration in vivo. The sample to be analyzed by the methods of theinvention may be contained in vivo, for instance in the measurement ofligand transport on the surface of cells, or in vitro, wherein ligandefflux) may be measured in cell culture. Alternatively, a fluid extractfrom cells or tissues may be used as a sample from which ligands aredetected or measured.

Methods for detecting ligands as disclosed herein may be used to screenand identify compounds that may be used to modulate ligand receptorbinding. In one embodiment, among others, the invention comprises amethod of identifying a compound that modulates binding of a ligand to areceptor, comprising (a) contacting a mixture comprising a cellexpressing a biosensor nucleic acid of the present invention and saidligand with one or more test compounds; and (b) determining FRET betweensaid donor fluorescent domain and said acceptor fluorescent domainfollowing said contacting, wherein increased or decreased FRET followingsaid contacting indicates that said test compound is a compound thatmodulates ligand binding. The term “modulate” generally means that suchcompounds may increase or decrease or inhibit the interaction of aligand with the ligand binding domain.

The methods of the present invention may also be used as a tool for highthroughput and high content drug screening. For instance, a solidsupport or multiwell dish comprising the biosensors of the presentinvention may be used to screen multiple potential drug candidatessimultaneously. Thus, the invention comprises a high throughput methodof identifying compounds that modulate binding of a ligand to areceptor, comprising (a) contacting a solid support comprising at leastone biosensor of the present invention, or at least one cell expressinga biosensor nucleic acid of the present invention, with said ligand anda plurality of test compounds; and (b) determining FRET between saiddonor fluorescent domain and said acceptor fluorescent domain followingsaid contacting, wherein increased or decreased FRET following saidcontacting indicates that a particular test compound is a compound thatmodulates ligand binding.

The targeting of the sensor to the outer leaflet of the plasma membraneis only one embodiment of the potential applications. It demonstratesthat the nanosensor can be targeted to a specific compartment.Alternatively, other targeting sequences may be used to express thesensors in other compartments such as vesicles, ER, vacuole, etc.

It is possible to use the sensors as tools to modify ligand binding, forinstance, by introducing them as artificial ligand scavengers presentedon membrane or artificial lipid complexes. Artificial ligand scavengersmay be used to manipulate signal transduction and the response of cellsto various ligands.

The following examples are provided to describe and illustrate thepresent invention. As such, they should not be construed to limit thescope of the invention. Those in the art will well appreciate that manyother embodiments also fall within the scope of the invention, as it isdescribed hereinabove and in the claims.

EXAMPLES Example 1 Use of Plants Suppressed in Gene Silencing PreventsSilencing of Direct Repeat Transgene

Repeated attempts to express biosensor transgenes in planta led to lowor no stable expression. Several independent attempts to generate plantsstably expressing biosensors for glucose, maltose and glutamate were notsuccessful, and resulted in either no expression at all or onlyexpression in young plants or expression only in guard cells. However,high expression in all tissues throughout plant development is desired.

Upon encountering difficulty in expressing the periplasmic bindingprotein-based biosensors in plants, the present inventors hypothesizedthat gene silencing in plants was affecting the expression of thetransgene constructs via repeat-induced silencing. The biosensors usedcontain eCFP and eYFP attached to the two ends of a substrate bindingprotein (FIG. 1). eCFP and eYFP are highly homologous, with only 9 outof 239 amino acids differing on the protein level and 16 out of 720 basepairs differing on the nucleic acid level (FIG. 2A). The use of eYFPVenus (Nagai et al., 2002, A variant of yellow fluorescent protein withfast and efficient maturation for cell biological applications, Nat.Biotech. 20: 87-90) leads to even higher homology, with only 8 aminoacids difference at the protein level and 13 base pairs difference atthe DNA level (FIG. 2B).

Two Arabidopsis genes, SGS3 and RDR6, have been described as beingrequired for posttranscriptional gene silencing. Peragine et al., 2004,SGS3 and SGS2/SDE1/RDR6 are required for juvenile development and theproduction of transacting siRNAs in Arabidopsis, Genes and Dev. 18:2368-79. To test our hypothesis, loss of function mutants for thesegenes) and Col0 wold type plants were transformed in parallel with theglucose sensor FLIPgludelta 13 (FIG. 3; Deuschle et al., 2005,Construction and optimization of a family of genetically encodedmetabolite sensors by semirational protein engineering, Protein Sci.14:2304-14). sgs3-11 plants were transformed with FLIPglu2μdelta13,rdr6-11 plants were transformed with FLIPglu600μdelta13, and Col0 plantswere transformed with FLIPglu2μdelta13 or FLIPglu600μdelta13. For alltransformations, the binary vector pPZP312 conferring Basta resistanceto transformed plants was used.

Transformants for two different affinity mutants of FLIPgludelta13 (2μand 600μ) were selected by spraying the seedlings of T1 with BASTA andscreened for fluorescence. A higher proportion of the transformants inthe sgs3-11 and rdr6-11 mutant background showed fluorescence than inthe Col0 background. The fluorescence of the Col0 transformants gotweaker with increasing plant age, whereas fluorescence in thesgs3-11/rdr6-11 transformants was at least detectable in plants at theonset of setting seeds (around 30 days after germination). Thisdifference in fluorescence intensity is not likely to be caused by adifferent number of T-DNA insertions, as segregation of the nextgeneration was around 3:1, suggesting a single insertion for most of thechecked plants.

Detection of changes in the cytosolic glucose level of plant cellscaused by external application of glucose was possible in rdr6-11 plantsexpressing FLIPglu600μdelta13 (see FIG. 4). As expected, no cytosolicglucose changes could be observed in sgs3-11 plants expressingFLIPglu2μdelta13, which is most likely saturated in the cytosol of plantcells.

Example 2 Decreasing the Homology of Repeat Sequences in Biosensors

In the genetic code, most amino acid sequences are encoded by more thanone codon. Exploiting this redundancy, genes can be synthesized usingdifferent codons than the original sequence, but still encoding the sameamino acid sequence. By changing the codon usage for at least one of thepartners of a tandem repeat, the percentage of homology can besignificantly decreased.

To circumvent gene silencing of the biosensor constructs, the homologyof the eCFP and Venus genes was decreased. To accomplish this, genesencoding a shortened eCFP (amino acids 7-230) and a shortened Venus(amino acids 7-230), each containing different codons with respect toeach other while keeping the same amino acid sequences of eCFP andVenus, were synthesized chemically. Shortened versions were synthesizedto save on synthesis costs. For cloning into expression vectorconstructs, the shortened versions may be amplified with extensionprimers to add back in the terminal sequences, which may also bedesigned with degenerate substitutions if desired. Alternatively, theshorter versions themselves may be used, as we have found that in somecases the closer coupling of the fluorophores can lead to higher ratiochanges upon ligand binding.

The genetically altered eCFP and Venus sequences were named Ares andAphrodite, respectively. Roughly every second codon was replaced in eachsequence, in an alternating pattern between the two genes. The newsequences differ in 228 out of 672 base pairs, and exclude identicalstretches longer than five base pairs (FIG. 5A). If only Venus isreplaced by Aphrodite, the longest stretch identical to eCFP is 11 basepairs (FIG. 5B).

Ares and Aphrodite were used as a FRET pair in FLIPglu600μdelta11(Deuschle et al., 2005) and successfully expressed in E. coli.Expression of Aphrodite could be shown in plants, where fluorophoreexpression was visibly enhanced as compared to the eYFP derivative (FIG.6). Thus, it appears that expression of Ares and Aphrodite in plantsshould circumvent or at least decrease homology dependent genesilencing. A shortened version of Venus in which nearly every codon wasmodified was also synthesized and named Mars (SEQ ID NO: 6). Mars isfunctional as a FRET partner of eCFP in vitro and can be expressed in E.coli. However, Mars has a significantly lower GC content than Aphrodite,which may lead to less than optimal expression in plants.

All publications, patents and patent applications discussed herein areincorporated herein by reference. While the invention has been describedin connection with specific embodiments thereof, it will be understoodthat it is capable of further modifications and this application isintended to cover any variations, uses, or adaptations of the inventionfollowing, in general, the principles of the invention and includingsuch departures from the present disclosure as come within known orcustomary practice within the art to which the invention pertains and asmay be applied to the essential features hereinbefore set forth and asfollows in the scope of the appended claims.

1. An isolated nucleic acid which encodes a ligand binding fluorescentindicator, the indicator comprising: a ligand binding protein moiety; adonor fluorophore moiety fused to the ligand binding protein moiety; andan acceptor fluorophore moiety fused to the ligand binding proteinmoiety; wherein fluorescence resonance energy transfer (FRET) betweenthe donor moiety and the acceptor moiety is altered when the donormoiety is excited and said ligand binds to the ligand binding proteinmoiety, and wherein the nucleic acid sequence encoding at least one ofeither said donor fluorophore moiety or said acceptor fluorophore moietyhas been genetically altered to reduce the level of nucleic acidsequence identity between the nucleic acid encoding the donorfluorophore moiety and the nucleic acid encoding the acceptorfluorophore moiety.
 2. The isolated nucleic acid of claim 1, wherein thenucleic acid sequences encoding both of said donor fluorophore moietyand said acceptor fluorophore moiety have been genetically altered toreduce the level of nucleic acid sequence identity between the nucleicacid encoding the donor fluorophore moiety and the nucleic acid encodingthe acceptor fluorophore moiety.
 3. The isolated nucleic acid of claim1, wherein said genetic alterations do not change the emission orabsorption spectra of said donor fluorophore moiety and said acceptorfluorophore moiety, respectively.
 4. The isolated nucleic acid of claim3, wherein said genetic alterations encode at least one conservativesubstitution in said donor or acceptor fluorophore.
 5. The isolatednucleic acid of claim 3, wherein said genetic alterations comprise atleast one degenerate substitution at a wobble position of the donor oracceptor fluorophore coding sequence.
 6. The isolated nucleic acid ofclaim 1, wherein said encoded ligand binding fluorescent indicatordemonstrates enhanced function in vivo upon expression of said nucleicacid containing said genetic alterations as compared to said ligandbinding fluorescent indicator expressed from said nucleic acid in theabsence of said genetic alterations.
 7. The isolated nucleic acid ofclaim 6, wherein said enhanced in vivo function occurs in a plant,animal or fungi.
 8. The isolated nucleic acid of claim 6, wherein saidenhanced in vivo function is due to a decrease in gene silencing.
 9. Theisolated nucleic acid of claim 1, wherein said donor and acceptorfluorophore moieties are fused to N- and C-termini of said ligandbinding moiety.
 10. The isolated nucleic acid of claim 1, wherein atleast one of either said donor fluorophore moiety or said acceptorfluorophore moiety is fused to said ligand binding protein moiety at aninternal site of said ligand binding protein moiety.
 11. The isolatednucleic acid of claim 1, wherein both said donor fluorophore moiety andsaid acceptor fluorophore moiety are fused to internal sites of saidligand binding protein moiety.
 12. The isolated nucleic acid of claim 1,wherein said ligand binding protein moiety is a transporter.
 13. Theisolated nucleic acid molecule of claim 12, wherein said transporter isselected from the group consisting of channels, uniporters, coportersand antiporters.
 14. The isolated nucleic acid of claim 1, wherein saidligand binding protein moiety is a periplasmic binding protein (PBP).15. The isolated nucleic acid of claim 14, wherein said ligand bindingprotein moiety is a bacterial periplasmic binding protein.
 16. Theisolated nucleic acid of claim 14, wherein said donor fluorescent moietyand said acceptor fluorescent moiety are fused to the same lobe of saidPBP.
 17. The isolated nucleic acid of claim 1, wherein said ligand is anamino acid.
 18. The isolated nucleic acid of claim 17, wherein saidamino acid is selected from the group consisting of glutamate,aspartate, γ-aminobutyric acid (GABA), aminoacetic acid (glycine) andtaurine.
 19. The isolated nucleic acid of claim 1, wherein said ligandis a sugar.
 20. The isolated nucleic acid of claim 19, wherein saidsugar is selected from the group consisting of glucose, galactose,maltose, sucrose, trehalose, arabinose, fructose, xylose, cellobiose andribose.
 21. The isolated nucleic acid of claim 1, wherein said donorfluorophore is selected from the group consisting of a GFP, a CFP, aBFP, a YFP, a dsRED, CoralHue Midoriishi-Cyan (MiCy) and monomericCoralHue Kusabira-Orange (mKO).
 22. The isolated nucleic acid of claim1, wherein said acceptor fluorophore moiety is selected from the groupconsisting of a GFP, a CFP, a BFP, a YFP, a dsRED, CoralHueMidoriishi-Cyan (MiCy) and monomeric CoralHue Kusabira-Orange (mKO). 23.The isolated nucleic acid of claim 21, wherein said donor fluorophoremoiety is a genetically altered version of eCFP.
 24. The isolatednucleic acid of claim 23, wherein said donor fluorophore moiety nucleicacid sequence contains the sequence SEQ ID NO: 1 (Ares).
 25. Theisolated nucleic acid of claim 1, wherein said acceptor fluorophoremoiety is a genetically altered version of YFP VENUS.
 26. The isolatednucleic acid of claim 25, wherein said donor fluorophore moiety nucleicacid sequence contains the sequence SEQ ID NO: 2 (Aphrodite).
 27. A cellexpressing the nucleic acid of claim
 1. 28. An expression vectorcomprising the nucleic acid of claim
 1. 29. A cell comprising the vectorof claim
 28. 30. The expression vector of claim 28 adapted for functionin a prokaryotic cell.
 31. The expression vector of claim 28 adapted forfunction in a eukaryotic cell.
 32. The cell of claim 29, wherein thecell is a prokaryote.
 33. The cell of claim 32, wherein the cell is E.coli.
 34. The cell of claim 25, wherein the cell is a eukaryotic cell.35. The cell of claim 34, wherein the cell is a yeast cell.
 36. The cellof claim 34, wherein the cell is an animal cell.
 37. The cell of claim34, wherein said cell is a plant cell.
 38. A transgenic animalexpressing the nucleic acid of claim
 1. 39. A transgenic plantexpressing the nucleic acid of claim
 1. 40. The isolated nucleic acid ofclaim 1, further comprising one or more nucleic acid alterations thatmodify the affinity of the ligand binding protein moiety to said ligand.41. A ligand binding fluorescent indicator encoded by the nucleic acidof claim
 1. 42. A method of detecting changes in the level of a ligandin a sample, comprising: (a) providing a cell expressing the nucleicacid of claim 1 and a sample comprising said ligand; and (b) detecting achange in FRET between said donor fluorophore moiety and said acceptorfluorophore moiety, wherein a change in FRET between said donor moietyand said acceptor moiety indicates a change in the level of said ligandin the sample.
 43. The method of claim 42, wherein the step ofdetermining FRET comprises measuring light emitted from the acceptorfluorophore moiety.
 44. The method of claim 42, wherein determining FRETcomprises measuring light emitted from the donor fluorophore moiety,measuring light emitted from the acceptor fluorophore moiety, andcalculating a ratio of the light emitted from the donor fluorophoremoiety and the light emitted from the acceptor fluorophore moiety. 45.The method of claim 42, wherein the step of determining FRET comprisesmeasuring the excited state lifetime of the donor moiety.
 46. The methodof claim 42, wherein said cell is contained in vivo.
 47. The method ofclaim 42, wherein said cell is contained in vitro.
 48. The method ofclaim 42, wherein fluorescence resonance energy transfer (FRET) betweenthe donor moiety and the acceptor moiety is increased when the donormoiety is excited and said ligand binds to the ligand binding proteinmoiety.
 49. The method of claim 42, wherein fluorescence resonanceenergy transfer (FRET) between the donor moiety and the acceptor moietyis decreased when the donor moiety is excited and said ligand binds tothe ligand binding protein moiety.
 50. An isolated nucleic acid whichcomprises a genetically modified fluorophore coding sequence, whereinsaid genetically modified fluorophore coding sequence contains at leastone wobble position base substitution as compared to the fluorophorecoding sequence that has not been genetically modified.
 51. The isolatednucleic acid of claim 50, wherein said genetically modified fluorophorecoding sequence contains at least two wobble position base substitutionsas compared to the fluorophore coding sequence that has not beengenetically modified.
 52. The isolated nucleic acid of claim 50, whereinsaid genetically modified fluorophore coding sequence contains at leastfive wobble position base substitutions as compared to the fluorophorecoding sequence that has not been genetically modified.
 53. The isolatednucleic acid of claim 50, wherein said genetically modified fluorophorecoding sequence contains at least ten wobble position base substitutionsas compared to the fluorophore coding sequence that has not beengenetically modified.
 54. The isolated nucleic acid of claim 50, whereinsaid genetically modified fluorophore coding sequence contains at leastfifteen wobble position base substitutions as compared to thefluorophore coding sequence that has not been genetically modified. 55.The isolated nucleic acid of claim 50, wherein said genetically modifiedfluorophore coding sequence contains at least twenty wobble positionbase substitutions as compared to the fluorophore coding sequence thathas not been genetically modified.
 56. The isolated nucleic acid ofclaim 50, wherein said genetically modified fluorophore coding sequencecontains at least thirty wobble position base substitutions as comparedto the fluorophore coding sequence that has not been geneticallymodified.
 57. The isolated nucleic acid of claim 50, wherein saidgenetically modified fluorophore coding sequence contains at least fiftywobble position base substitutions as compared to the fluorophore codingsequence that has not been genetically modified.
 58. The isolatednucleic acid of claim 50, wherein said genetically modified fluorophorecoding sequence contains at least one hundred wobble position basesubstitutions as compared to the fluorophore coding sequence that hasnot been genetically modified.
 59. The isolated nucleic acid of claim50, wherein said fluorophore is a genetically modified version of eCFP.60. The isolated nucleic acid of claim 59, wherein said fluorophorenucleic acid sequence contains the sequence SEQ ID NO: 1 (Ares).
 61. Theisolated nucleic acid of claim 50, wherein said fluorophore is agenetically modified version of YFP VENUS.
 62. The isolated nucleic acidof claim 61, wherein said fluorophore nucleic acid sequence contains thesequence SEQ ID NO: 2 (Aphrodite).
 63. A method of reducing genesilencing of one or more transgenes in a cell, comprising introducing atleast one genetic alteration into said one or more transgenes such thatthe level of identity in at least one repeat region of said one or moretransgenes is reduced, and transfecting said one or more transgenes intosaid cell, wherein gene silencing of said one or more transgenes isthere by reduced.
 64. The method of claim 63, wherein at least tworepeat regions are present a single transgene.
 65. The method of claim63, wherein said at least one repeat region is present in two or moredifferent transgenes.
 66. The method of claim 63, wherein said at leastone repeat region is present in said one or more transgenes and anotherrepeat region is within the DNA of said cell.
 67. The method of claim64, wherein said single transgene is a ligand binding fluorescentindicator comprising a ligand binding protein moiety, a donorfluorophore moiety fused to the ligand binding protein moiety; and anacceptor fluorophore moiety fused to the ligand binding protein moiety.68. The method of claim 64, wherein said single transgene encodes anartificial single chain dimer.
 69. The method of claim 64, wherein saidsingle transgene encodes a protein with duplicated domains (e.g., ABCtransporters).
 70. The method of claim 65, wherein said two or moredifferent transgenes encode proteins with substantially similar domains.71. The method of claim 63, wherein said cell is a plant cell.
 72. Themethod of claim 63, wherein said cell is an animal cell.
 73. The methodof claim 63, wherein said cell is in a plant.
 74. The method of claim63, wherein said cell is in an animal.
 75. The method of claim 63,wherein said at least one genetic alteration does not adversely affectthe function of the protein encoded by said transgene.
 76. The method ofclaim 75, wherein said at least one genetic alteration encodes aconservative amino acid substitution in said transgene.
 77. The methodof claim 75, wherein said at least one genetic alteration is adegenerate substitution at a wobble position of said transgene.
 78. Themethod of claim 77, comprising introducing at least two degeneratesubstitutions at wobble positions of said transgene.
 79. The method ofclaim 77, comprising introducing at least five degenerate substitutionsat wobble positions of said transgene.
 80. The method of claim 77,comprising introducing at least ten degenerate substitutions at wobblepositions of said transgene.
 81. The method of claim 77, comprisingintroducing at least fifteen degenerate substitutions at wobblepositions of said transgene.
 82. The method of claim 77, comprisingintroducing at least twenty degenerate substitutions at wobble positionsof said transgene.
 83. The method of claim 77, comprising introducing atleast thirty degenerate substitutions at wobble positions of saidtransgene.
 84. The method of claim 77, comprising introducing at leastfifty degenerate substitutions at wobble positions of said transgene.85. The method of claim 77, comprising introducing at least one hundreddegenerate substitutions at wobble positions of said transgene.
 86. Themethod of claim 63, wherein said at least one genetic alteration doesnot lower the GC content of said transgene.
 87. The method of claim 63,wherein said gene silencing is selected from the group consisting ofrepeat-induced gene silencing (RIGS), repeat-induced point mutation(RIP), paramutation, ectopic trans-inactivation, co-suppression and RNAinterference.